Evaluation of dose equivalent using a tissue-equivalent ionization chamber and a Geiger-Müller dosimeter

Summary When detailed neutron energy spectrum data are lacking for a mixed field of neutrons and photons, it is permissible when estimating the dose equivalent to assume that the quality factor for the neutrons is 10. With this assumption, it is shown that the responses of a tissue-equivalent ionization chamber and a Geiger-Müller dosimeter can be used to obtain an acceptable approximation of the dose equivalent in the mixed field without requiring precise knowledge of the relative neutron sensitivity of the Geiger-Müller dosimeter.This investigation was supported by Contract EP-78-S-02-4733 from the Department of Energy to the Radiological Research Laboratory/Department of Radiology and by Grant No. CA13696 to the Cancer Center/Institute of Cancer Research, awarded by National Cancer Institute, DHEW     

Improved conditions for murine epidermal cell culture

Summary An improved method for cultivating newborn mouse epidermal cells has been developed that increases the longevity, epithelial nature and efficiency of cell-line establishment. The use of Super Medium, an enriched Waymouth's formulation, increased proliferation for long periods of time, as did incubation at 31°C rather than 37°C. The fetal bovine serum requirement was found to be reduced at the lower temperature. An increase in labeling indices was seen when epidermal growth factor (EGF) or the cyclic nucleotides were added and the presence of EGF receptors was determined. Of the prostaglandins (PG) examined, PGE₁ and PGE₂ produced the greatest increase in DNA synthesis. The PG precursors, arachidonic and 8,11,14-eicosatrienoic acid, were also greatly stimulatory. The use of a lethally irradiated 3T3 feeder layer at 31°C proved superior in maintenance of an epithelial morphology. Subculturable cell lines were established much more readily and reproducibly in carcinogen-treated cultures grown under the improved conditions. Research sponsored jointly by the National Cancer Institute under Interagency Agreement YO1-CP-70227 and the Office of Health and Environmental Research, U.S. Department of Energy, under contract W-7405-eng-26 with the Union Carbide Corporation. Predoctoral Investigator supported by Grant CA 09104 from the National Cancer Institute. Postdoctoral Investigator supported by Carcinogenesis Training Grant CA 05296 from the National Cancer Institute.     

Characterization of cells cultured from early lactation milks

Summary Two major types of cells can be cultured from early lactation human milks: a colony-forming epithelial cell and an adherent nondividing cell referred to as a foam cell The epithelial cells show a positive reaction with a specific antiserum reactive against membrane components of the milk fat globule, whereas the foam cells do not. The nondividing foam cells are phagocytic and can be killed by silica particles; they produce lysozyme, are resistant to trypsinization, and have Fc receptors. These properties, together with the lack of reaction with antiserum to the milk fat globule membrane, suggest that the foam cells are not terminally differential epithelial cells, but tissue macrophages. R. L. C. was supported by Grant No. Ca 19455 from the National Cancer Institute, a Yamagiawa-Yoshida Memorial International Cancer Study Grant, and the Imperial Cancer Research Fund. J. A. P. was supported by Grant No. CA 19455 from the National Cancer Institute.     

Genetic and biochemical studies on the suppression of and a recovery from the tumorous state in higher plants

Summary Four neoplastic diseases of plants: crown gall, which is caused by Ti plasmid DNA; Black's wound tumor disease by an RNA virus; the Kostoff genetic tumors by chromosomal imbalance; and habituation, which results from a spontaneous activation of select biosynthetic systems, have been analyzed and compared. It has been found that both the development of a capacity for autonomous growth and the nature of the heritable cellular change that underlies tumorigenesis are similar in the four instances. All develop a capacity for autonomous growth as a result of the persistent activation of select biosynthetic systems, the products of which are concerned with cell growth and division. That the persistent activation of these biosynthetic systems does not involve heritable changes of an irreversible type is indicated by the finding that a reversal of the neoplastic state occurred in three of the test systems. Since the tumor cells in these instances were found to remain totipotent the results suggest that whether the normal or tumor phenotype is expressed is determined by how the genetic information is regulated in a cell. Regulation appears to be accomplished in part through positive feedback control mechanisms. Foreign genetic information could act either in a regulatory manner to persistently activate normal biosynthetic systems or it could code for one or more essential but normally limiting substance(s) and thus replace a substance(s) that in the case of the Kostoff tumors or habituation is specified by host cell genes, or it could do both. In either case, the foreign genetic information can be regulated in much the same manner as are the host cell genes to give rise to either the normal or tumor phenotype. Presented in the symposium on Gene Transfer, Differentiation and Neoplasia in Plant and Animal Cells at the 30th Annual Meeting of the Tissue Culture Association, Seattle, Washington, June 10–14, 1979. This symposium was supported in part by Grant CA 26748 from the National Cancer Institute, DHEW, and Grant RD-67 from the American Cancer Society. Certain of the investigations described above were supported in part by Grant Number CA-13808, awarded by the National Cancer Institute, U.S. Department of Health, Education, and Welfare, in which the author is the coprincipal investigator.     

Keratinization and the effect of vitamin A in aggregates of a squamous carcinoma cell line NBT II

Summary The terminal differentiation, keratinization, of a rat bladder tumor cell line, NBT II, occurred in multicellular aggregates. After aggregation, these cells did not undergo a round of mitosis before keratinization. 5-Bromodeoxyuridine added to the monolayer cell culture 2 days before aggregation completely prevented this differentiation; it was ineffective when added at the time of cell aggregation. Vitamin A prevented the keratinization of NBT II cells in aggregates but did not inhibit aggregate formation; it enhanced the number of cells engaged in DNA synthesis. This model appears to be very useful for analyzing the mechanisms of terminal differentiation and its modulation by vitamin A in tumor cells. This research was supported by Institutional Research Grant 731-01-E from the American Cancer Society and in part by Research Grant CA 14137 from the National Cancer Institute to Dr. J. Leighton.     

Histochemical identification of cultured cells from human endometrium

Summary Histochemical techniques have been applied to the identification of cell types cultured from human endometrium. Previous work from this laboratory characterized two principtal cell types found in cultures of endometrium: a mature epithelial cell and another cell which was classified as the endometrial stromal cell based on light and electron microscopy. In this report we compare the histochemical staining of endometrial tissue in frozen sections to that of cultured cells. These results confirm the epithelial and stromal nature of the respective cell types. Several markers were found that could distinguish between cells of epithelial and stromal origin. The enzymes alkaline phosphatase, γ-glutamyltranspeptidase, peroxidase, and β-glucuronidase were localized in glandular and surface epithelia in frozen sections and in colonies of epithelial cells in culture. Stroma in frozen sections and cultured stromal cells contained leucine aminopeptidase and fibronectin. Epithelial sections and in culture could also be distinguished from cells of stromal origin by preferential binding of lotus and peanut lectin. Several other markers were found in both endometrial epithelium and stroma. J. M. S. was recipient of National Research Service Award CA09156 (National Cancer Institute); K. G. N. was recipient of National Research Service Award ES07017 (National Institute of Environmental Health Sciences); and D. G. K. was recipient of Research Career Development Award CA00431 from the National Cancer Institute, Bethesda, MD. Supported by Grant CA 31733 from the National Cancer Institute, Bethesda, MD.     

Identification of mouse mammary adipose cells by membrane antigens

Summary Antisera produced to mammary adipose cells from midpregnant BALB/c females can be used to distinguish mammary adipose cells from mammary epithelial cells and fibroblast. The mammary adipose membrane antigen detected by indirect immunofluorescence was found in adipose cells from (a) mammary glands of virgin, midpregnant and lactating mice; (b) mammary fat pads that had been surgically cleared of glandular elements; and (c) epididymis. In all tissues, this cell-surface antigen was removed by the enzymatic action used to dissociate the cells from the tissues and was shown to be fully restored when cells were cultured for 48 hr. This work was supported by National Cancer Institute Grant Nos. CA11736 and CA18946 and Biomedical Research Support Grant No. RR05467 from the National Institutes of Health, DHEW.     

Quantitation of human mammary epithelial antigens in cells cultured from normal and cancerous breast tissues

Summary A sensitive radioimmunoassay technique was developed to quantitatite the level of human breast celltype specific antigens on cells from normal breast and from various established cell lines of breast and nonbreast origins. Polyacrylamide gel electrophoresis revealed four major proteinaceous components (150,000; 75,000; 60,000; and 48,000) in human milk fat globule membranes that were used to immunize rabbits in order to elicit antimammary epithelial cell antibody. Antisera obtained were rendered specific by abosorptions and were able to recognize three specific mammary epithelial components of the breast epithelial cell. Human mammary epithelial (HME) antigen expression was highest (1290 ng/10⁶ cells) in normal breast epithelial cells from primary cultures of normal breasts. Lower levels (range: 955 to 330 ng/10⁶ cells) were found in breast epithelial cells from cell lines established from cancerous breast tissue. Cells of nonbreast origins as well as fibroblasts from breast gave much lower values (less than 30 ng/10⁶ cells). On treatment, with trypsin, of two breast epithelial cell lines (MDA-MB-157 and MCF-7) 80 to 85% of their HME antigen expression was lost, suggesting that a majority of these breast antigens reside on the cell surface. This work was Supported by Grant PTD-99 from the American Cancer Society, Grant CA19455 and CA20286 from the National Cancer Institute, and Biomedical Research Support Grant RR05467 from the National Institutes of Health. Most cells used in the present study were produced with support from National Cancer Institute Contract Y01-CP8-0500, Biological Carcinogenesis Branch, Division of Cancer Cause and Prevention, under the auspices of the Office of Naval Research and the Regents of the University of California.     

The role of microdosimetry in radiobiology

Summary Microdosimetry is a branch of radiological physics that provides quantitative characterization of the non-uniformity of energy deposition in uniformly irradiated matter.Considerations based on microdosimetry indicate that the action of ionizing radiation on the cells of higher organisms depends on the square of the specific energy absorbed in subcellular volumes. This is the basis of the Theory of Dual Radiation Action. The basic postulate of this theory is reviewed and four factors are discussed that modify its elementary formulation.Presented at the 6th European Conference on Radiobiology (Julich 1978). Based on work carried out under Contract EP-78-S-02-4733 from the U.S. Department of Energy and by Grant Nos. CA 12536, CA 15307 of the National Cancer Institute     

Elucidation of an a and L system for amino acid transport in the human lymphoblast using a membrane filtration technique

Summary Optimum conditions have been established for the measurement of amino acid transport by human lymphoblastoid cell lines using a membrane-filtration technique. The parameters we found to be important for the reproducibility of the method are: the types and combination of filters, the strength of the vacuum applied to the filters and the density of the cultures at the time of harvesting and during uptake and filtration. We found that bovine serum albumin added to phosphate buffered saline (PBS) glucose in which the cells are washed, resuspended and assayed is essential for the maintenance of viability, the prevention of clumping and the retention of the accumulated amino acid. Using this procedure we have characterized two transport systems for the neutral amino acids; an A and an L system, which are similar but not identical to the A and L systems characterized in rodent cell lines. These A and L systems have characteristically lower Km's and Vm's for alanine and phenylalanine, when compared to rodent cell lines. In addition, we find α-AIB to be a poor competitor of alanine and phenylalanine uptake. This work was supported by Grant No. CA18644, awarded by the National Cancer Institute, Department of Health, Education and Welfare, and from a grant from the National Science Foundation under Grant No. PCM 76-24328.     

The use of bone core biopsies for cytogenetic analysis

Summary Cultures of bone core specimens have proved satisfactory for cytogenetic analysis in patients from whom it was impossible to obtain a bone marrow aspirate, or in whose peripheral blood dividing myeloid cells were absent or insufficient in number. The quality of the metaphase chromosome is adequate for banding studies.Supported by NIH Grant CA 16910 and an Otho S.A. Sprague institutional grant. The Franklin McLean Memorial Research Institute is operated by The University of Chicago for the United States Department of Energy under Contract EY-76-C02-0069     

Characteristics of rat normal mammary epithelial cells and dimethylbenzanthracene-induced mammary adenocarcinoma cells grown in monolayer culture

Summary The characteristics of normal mammary epithelial and 7,12-dimethylbenz[a]anthracene (DMBA)-induced adenocarcinoma cells derived from rats and grown in monolayer culture were compared. Normal mammary epithelial cells exhibited different morphology and agglutinability by plant lectins, slower growth rate, and lower saturation density and cloning efficiency. In addition, the normal cells were sensitive to the toxic effect of DMBA, and were unable to grow in soft agar or to form tumors, when inoculated into newborn Sparague-Dawley rats. The converse was true in each case for the adenocarcinoma cells. Supported by Public Health Service Research Grant CA 01237603 from the National Cancer Institute Portions of this paper were presented at the 65th Annual Meeting of the American Association for Cancer Research at Houston, Texas, 1974.     

Density-induced down regulation of epidermal growth factor receptors

Summary Previous studies have shown that cell density can regulate the binding of several growth factors. To determine whether cell density exerts a uniform effect on the expression of epidermal growth factor (EGF) receptors, seven cell lines were examined in detail. For each cell line, EGF binding was found to decrease as cell density increases. Scatchard analysis of the binding data reveals that decreases in EGF binding are due to reductions in the number of cell surface EGF receptors. The only apparent exception is the effect of cell density on the binding of EGF to A-431 cells. For these cells, increases in cell density lead to two effects: decreases in the number of high affinity EGF receptors and increases in the total number of EGF receptors. In addition to the effects of cell density on EGF receptors, it was determined that increases in cell density can coordinately down-regulate receptors for as many as four different growth factors. Overall, the findings described in this report for EGF and those previously described for transforming growth factor type-β (TGF-β) and fibroblast growth factor (FGF) demonstrate the existence of a common mechanism for down-regulating growth factor receptors. This work was supported by grants from the Nebraska Department of Health (89-51), the National Cancer Institute (Laboratory Research Center Support Grant, CA36727), and the American Cancer Society (Core Grant ACS SIG-16). EDITOR'S STATEMENT The paper by Rizzino et al. demonstrates that receptor number decreases as a function of cell density. This may represent a mechanism by which cell proliferation is reduced as cell density increases.     

Ducts of the rat pancreas in agarose matrix culture

Summary Interlobular and intralobular ducts isolated from the pancreas of the rat by digestion with collagenase and chymotrypsin were cultured in an agarose matrix containing CMRL-1066 supplemented with insulin, dexamethasone,l-glutamine, soybean trypsin inhibitor, antibiotics, and fetal bovine serum. The cut ends of most interlobular ducts sealed to create encolosed lumina. Some ducts retained their original cylindrical organization; others enlarged to varying degrees, resulting in structures that ranged from cylindrical to spherical in shape. The duct walls consisted of viable epithelium and connective tissue, although the amount of connective tissue declined with age. Both epithelial and connective tissue cells became flattened in the enlarged ducts. Intralobular and small interlobular ducts often remained associated with the larger interlobular ducts. These duct fragments have been cultured for as long as 6 weeks. This study was supported by National Cancer Institute Grant CA 19177 through the National Pancreatic Cancer Project and by Biomedical Research Support Grant RR 07196 from the Division of Research Resources, National Institutes of Health.     

Cell transformation by viruses

Summary This paper describes some current work pertaining to transformation of cells by oncogenic viruses. Part I includes: (1) the effect of a deoxyribonucleic acid (DNA) tumor virus (SV40) on the antigenic characteristics of transformed cells; (2) in vitro and in vivo methods of detecting virus-specific surface antigens; (3) the role that the host cell may play in the expression of virus-coded antigens; and (4) the presence of virus-induced antigens as a possible mechanism of the apparent nononcogenicity of certain virus variants. Part II discusses (1) the physicochemical properties of the nucleic acid of a ribonucleic acid (RNA) tumor virus-the Moloney sarcoma-leukemia virus (MSV-MLV) complex —(2) a preliminary analysis of viral RNA replication in cells transformed by MSV-MLV, and (3) application to human tumors. Supported in part by Research Grant CA 04600 and by Research Contract PH 43-68-678 within the Special Virus-Cancer Program, National Cancer Institute, National Institutes of Health. Recipient of Research Career Development Award 5-K3-CA 38,614 from the National Cancer Institute, National Institutes of Health     

Presence of retrovirus in the B95-8 Epstein-Barr virus-producing cell line from different sources

Summary The B95-8 cell line, a widely used source of highly transforming Epstein-Barr virus (EBV), obtained from the laboratory of origin, harbored an infectious retrovirus. This retrovirus generally resembled the Type D retroviruses structurally and developmentally and like the Type D retroviruses preferred Mg²⁺ to Mn²⁺ in its RNA-directed DNA polymerase reaction. Evidence for the presence of retrovirus was found in B95-8 cultures from two other sources within the United States, either by assay for polymerase or by electron microscopy. Comparison of two B95-8 cell lines showed cytogenetic differences as well as differences in retroviral activities. The results suggest that any B95-8 culture should be tested for the presence of retrovirus before its use as a source of EBV. This research was supported through the National Research and Demonstration Center (HL-17269-07) awarded to Baylor College of Medicine by the National Heart, Lung, and Blood Institute, Bethesda, MD, by RD-125 from the American Cancer Society, by K06 CA14219, CA16781, CA25465, and CA16672 from the National Cancer Institute, Bethesda, MD, and by G-429 from the Robert A. Welch Foundation. G. E. G. was supported by Public Health Service training Grant CA-09299.     

A simple liquid medium for chlamydospore formation in Candida albicans

A new, relatively simple and inexpensive liquid medium was devised to produce all structural forms ofC. albicans. Optimum conditions to induce the yeast cells, germ tubes, pseudohyphae and chlamydospores along with the methods to obtain them are described.Supported in part by Grant CA 20917, National Cancer Institute, National Institutes of Health and ALSAC.     

Methods for the study ofin vitro immunization of mouse spleen cells

Conclusion We have reviewed some of our experiences in developing techniques for studying the functions of the cells of the immune system. It is quite clear that much remains to be done. Improvements in the culture system are needed to permit cells to be grown for longer durations and at lower cell concentrations. The important effects of fetal calf serum should be defined. More sophisticated methods for separating cells into distinct functional populations must be developed. New assays for identifying other functions of the cells, particularly a method for directly assaying the number of precursor cells in a population, are needed. When these techniques are applied to the study of immune cells, further facts should be learned which will permit the development of significant, testable hypotheses on the function and relationships of the cells of the immune system. This is publication No. 298 from the Department of Experimental Pathology, Scripps Clinic and Research Foundation, La Jolla, California 92037. This work was supported in part by U.S.P.H.S. Grant 7007 and in part by American Cancer Society Grant E-395. Dr. Mishell is supported by American Cancer Society Grant E-395. Dr. Dutton is supported by a Dernham Fellowship of the California Division, American Cancer Society (No. D-100). Dr. Raidt is supported by United States Public Health Service Postdoctoral Fellowship No. 7-F2-A1-31,590.     

Fine structural identification of organoid mouse lung cells cultured on a pigskin substrate

Summary Mouse full-term embryonic lung tissue was cultured as organ bits using dead, sterile pigskin dermal collagen as a substrate. Explanted organ bits grew on the surface of, and into, the pigskin dermal collagen for at least 9 weeks after the initiation of culture. The out-growth consisted of a thick cellular sheet containing various sizes of ductular structures within a cellular matrix that did not show any particular structure. Electron microscopic observation revealed that the larger ductular structures consisted largely of ciliated cells. The smaller ductular structure consisted largely of Type II pneumocytes containing lamellar hodies. The cellular matrix consisted of Type II pneumonocytes and other cell types including fibroblasts and macrophages in the early stage of cultivation. Macrophages invaded the pigskin dermal collagen. An intermediate cell type, which has never been observed in vivo, possessing both cilia and lamellar bodies was identified in the larger ductular structures. Upon comparison of the ultrastructure of the organoid in vitro cultures in pigskin with the components and structure of the cultured cells more closely resembled adult lung than the fetal lung used to initiate the cultures. This work was supported by the Council for Tobacco Research Grant 1203M, American Cancer Society Grant RD-65 (for the equipment), and the National Cancer Institute Grant CA 25392.